We have shown that antigen-stimulated exocytosis in a mast cell line, RBL-2H3 cells is accompanied by an increased phosphorylation of both the heavy (200 kDa) and light (20 kDa) chains of myosin, by protein kinase C (PKC). Using an isoelectro-focusing technique, we were able to map out the sites of phosphorylation of 20 kDa myosin light chain. In unstimulated cells, the myosin light chains were already phosphorylated by myosin light chain kinase (MLCK) at a single site (serine 19). In antigen stimulated cells, there was de novo phosphorylation of two additional sites, one by PKC (serine 1) and one by MLCK (threonine-18). The phosphorylation by MLCK, which is a calcium-regulated enzyme, and by PKC, may be significant because exocytosis in RBL-2H3 cells is totally dependent on a rise in [Ca2+]i and the activation of PKC. Furthermore, these phosphorylations could be induced by Ca2+-ionophore A23187 and phorbol ester but only at concentrations that were sufficient to induce secretion. Also selective suppression of MLCK (with KT5926) or PKC (with Ro31-7549) inhibited secretion and the phosphorylation by these enzymes. Kinetic studies also revealed that the phosphorylation of myosin light chains by PKC and MLCK was rapid and correlated with secretion.An additional indication that phosphorylation by both kinases was necessary for secretion was that stimulation of cells via adenosine (A3) receptors resulted in sustained phosphorylation of the light chains by PKC but only transient elevation of [Ca2+]i, minimal phosphorylation of myosin light chains by MLCK, and no secretion.